Genetic complementation strategies have greatly facilitated the identification of regulatory genes in haploid organisms such as E. coli and Saccharomyces cerevisiae. The drawback of using such an approach in diploid mammalian systems has been the lack of mutant cell lines to act as recipients of DNA, and the limited number of selection techniques. To circumvent these problems, hybrid genes have been prepared which contain the transcriptional control regions of the mouse alpha-fetoprotein (AFP) gene linked to a structural gene encoding a cell surface mouse major histocompatibility complex (MHC) protein, providing a sensitive immunological assay for gene activation with MHC-specific monoclonal antibodies. AFP-nonexpressing mouse L cells are stably transfected with AFP-MHC hybrid genes and then used as recipients for a second round of transfection with human genomic DNA. Cells are screened to identify those which have activated the AFP-MHC genes presumably due to the transfer of a human regulatory gene, which is subsequently isolated using species- specific repetitive element DNA probes. A series of preliminary experiments established the feasibility of this approach. The first series of genomic DNA transfections resulted in the identification of one cell line which had activated AFP-MHC hybrid genes. Cells which have deleted the human DNA no longer express hybrid genes. This and other experiments argues that the human DNA is responsible for hybrid gene activation. Experiments are proposed to confirm that the genomic DNA is responsible for transactivation. The second objective is to improve the genetic strategy and use a cDNA library as a source of transactivators in order to facilitate efforts to identify other genes which regulate AFP expression. Structural and functional analyses of transactivating proteins will also be performed. The onset of many diseases, most notably cancer, is often the consequence of inappropriate gene expression. To understand this process better, it is important to determine the mechanism responsible for normal gene regulation.